Chimeric antigen receptor T cells for gamma–delta T cell malignancies

Abstract

Cancers derived from the malignant transformation of gamma–delta (γδ) T cells carry very poor prognosis. The major pathologies recognised are γδ T acute lymphoblastic leukaemia (γδ T-ALL), and two lymphoma subtypes: hepatosplenic T cell lymphoma (HSTL) and primary cutaneous γδ T cell lymphoma (PCγδ-TCL) [ 1 ]. γδ T-ALL represents approximately 10% of cases of T-ALL and is associated with high rates of induction failure, relapse and excess mortality [ 2 ]. HSTL is a rare (approximately 3% of cases of T cell lymphoma [ 1 ]) but highly aggressive disorder, which typically presents in males in the 2nd or 3rd decade of life, often in association with immunosuppressive therapy [ 3 ]. It carries the worst prognosis of all lymphoma subtypes, with a median survival of only 6–8 months [ 4 ] and only isolated cases of long-term survival [ 5 ]. PCγδ-TCL is also rare (approximately 1% of skin lymphomas [ 1 ]) and presents with cutaneous involvement, typically associated with visceral and/or bone marrow disease. Again, outcomes are poor, with 75% 1-year mortality in the largest published case series [ 6 ]. Treatment for γδ malignancies is with cytotoxic chemotherapy, with no tumour-specific therapies currently available. By contrast, in analogous B-cell malignancies, highly effective immunotherapies, including monoclonal antibodies, bispecific T cell engagers and chimeric antigen receptor (CAR)-T cells [ 7 ] are available. These therapies have revolutionised the treatment and outcome of advanced B-cell malignancies. CAR-T cells against CD19 in particular have demonstrated the potential to induce long-lasting complete remissions even in patients with advanced and refractory cancers [ 7 ]. For γδ malignancies, the defining immunophenotypic characteristic is expression of the γδ T cell receptor (TCR), present in >95% of cases of HSTL and in all PCγδ-TCL and γδ T-ALL [ 3 ]. Importantly, in normal tissues expression is limited to γδ T cells, where it functions as the antigen recognition receptor. Here, we developed CAR-T cells targeting the γδ TCR and demonstrate in vitro and in vivo efficacy against γδ T cell malignancies. Our data offers proof-of-concept for γδ TCR-targeting with CAR-T cells as a potential approach to bring highly potent immunotherapy to the treatment of γδ malignancies. Primary αβ T cells were retrovirally transduced to express anti-γδ TCR CAR or control anti-CD19 CAR (Fig. 1a ). Following transduction with anti-CD19 CAR, a small proportion of γδ T cells persisted in the culture, including some which expressed anti-CD19 CAR. By contrast, for anti-γδ TCR CAR, no γδ T cells were detected in the culture, suggesting ‘purging’ of these cells by the transduced population (Fig. 1b ). CAR-T cells were then co-cultured with T cell lines which natively express (Loucy – Vγ9Vδ2, BE13 – Vγ8Vδ1, MOLT13 – Vγ3Vδ1 [ 8 ]) or are negative for surface γδ TCR (SupT1-CD19). While control anti-CD19 CAR lysed only SupT1-CD19 cells, anti-γδ TCR CAR-T lysed only γδ TCR-positive cell lines (Fig. 1c ). In addition, anti-γδ TCR CAR-T cells demonstrated specific secretion of cytokines including interferon-γ, IL-2, IL-13 and TNF-α (Fig. 1d ). Next, we co-cultured anti-CD19 or anti-γδ CAR-T cells with normal autologous γδ T cells. At a high E:T ratio (1:1), target normal γδ T cells were partially lysed (Fig. 1e ), with concomitant expansion of anti-γδ CAR-T cells (Fig. 1f ). A marked downregulation of γδ TCR expression was noted on surviving γδ T cells (Fig. 1g ). Interestingly, by contrast, at lower E:T ratios (1:2 and 1:4), paradoxical γδ T cell expansion was instead observed (Fig. 1e ), associated with reduction in numbers of anti-γδ CAR-T cells (Fig. 1f ). This suggests lysis of anti-γδ CAR-T by target normal γδ T cells. Fig. 1: In vitro testing of anti-γδ TCR CAR. a Schematic of anti-γδ TCR CAR, with 2nd generation architecture. b Example flow plot of γδ-TCR staining on anti-CD19 or anti-γδ TCR CAR-T cells following transduction c co-cultures of anti-γδ TCR or control anti-CD19 CAR-T cells with CD19+ (SupT1-CD19) or γδ TCR+ cell lines (Loucy, MOLT13, BE13) ( c ) cytotoxicity at 72 h, as measured by bioluminescence-based assay ( d ) cytokine secretion at 48 h. e – g 120-h co-culture of control or anti-γδ TCR CAR-T cells with autologous normal γδ T cells, n = 3. e Residual γδ T cells as proportion of starting cells f Example γδ TCR staining on normal γδ T cells after co-culture with anti-γδ TCR CAR-T or anti-CD19 CAR-T cells g residual anti-γδ TCR CAR-T or anti-CD19 CAR-T following co-culture, as proportion of starting cells. ** p < 0.001, *** p < 0.0001. Full size image To assay the in vivo potency of anti-γδ TCR CAR-T cells, we utilised the Loucy murine model of disseminated γδ TCR-positive leukaemia (Fig. 2a, d ). NSG mice were intravenously injected on CAR D-12 with 4 × 10 6 Loucy cells, engineered to stably express Firefly luciferase. Tumour engraftment was confirmed by bioluminescence imaging (BLI) at D-1 (data not shown), then mice were treated on D0 with 8 × 10 5 anti-γδ TCR or control anti-CD19 CAR-T cells. Mice receiving anti-γδ TCR CAR demonstrated reduction of tumour burden, as assessed by flow cytometry of bone marrow and spleen at necropsy on D14 (Fig. 2b, c , Supplementary Fig. 1 ), BLI (Fig. 2e, f ) and bleed at D30 (Fig. 2g ). Prolonged survival (Fig. 2h ) was seen in anti-γδ TCR CAR recipients compared to CD19 CAR-treated animals, although all animals eventually died of progressive γδ TCR-positive disease, with no evidence of antigen downregulation. In common with other NSG models, CAR-T cell persistence was limited, with no detectable cells in the peripheral blood at D30 (data not shown). Fig. 2: In vivo assessment of anti-γδ TCR CAR. a Schematic of Loucy murine model ( n = 6/group) b Quantification of tumour in ( b ) marrow and ( c ) spleen at D14 following CAR-T injection. d Schematic of Loucy murine model (n = 9/group) e bioluminescence (BLI) imaging at D21 following CAR-T infusion. f Quantification of BLI signal at D21 g quantification of tumour burden in blood at D30 h survival curves of mice (median survival CAR19 50 days v CARγδ 69 days, HR 12.4, p = 0.0003, comparison by log-rank method). All other comparisons by Mann–Whitney U test. Full size image Despite success in B-cell malignancies, a lack of acceptable targets means targeted immunotherapy is rarely applied to T cell malignancies. The anti-CD30 antibody-drug conjugate brentuximab vedotin is effective in anaplastic large cell lymphoma [ 9 ], however, CD30 is not typically expressed in γδ cancers [ 2 ]. Suggested approaches to targeting T cell malignancies include targeting pan-T cell antigens such as CD5 [ 10 ] or CD7 [ 11 ]. However, such strategies deplete the entire normal T cell compartment and may induce profound immunosuppression [ 11 ], potentially requiring rescue by allogeneic hematopoietic stem cell transplant. More refined approaches that target clonal elements of the TCR, such as selective targeting of TRBC1 and TRBC2 in αβ T cell malignancies, allow depletion of only part of the normal T cell compartment [ 12 ]. While analogous approaches are potentially possible in γδ TCR malignancies, simple targeting of the γδ TCR may be feasible. This approach could concomitantly deplete normal γδ T cells. These constitute <5% of peripheral blood T cells, are more abundant in tissues and have a range of proposed ancillary immunological functions [ 13 ]. Importantly, genetically γδ-deficient mice display a very mild phenotype [ 14 ], and there is no known human pathology associated with γδ T cell deficiency. This suggests that depletion of the γδ T cell compartment may be clinically tolerable, although initially clinical exploration of anti-γδ TCR CAR-T should proceed cautiously: for instance with co-expression of a suicide gene [ 15 ], availability of back-up cryopreserved peripheral blood mononuclear cells, and close monitoring for development of atypical infections. Indeed, it is unclear if anti-γδ CAR-T treatment would lead to γδ T cell aplasia. An interesting observation in our study was that, while anti-γδ CAR-T expanded when cultured with normal γδ T cells at high E:T ratio, the reverse was observed when normal γδ T was in excess. Thus, anti-γδ CAR-T were themselves depleted from the culture, with expansion of normal γδ T cells. The probable explanation is that CAR binding to the TCR of γδ T cells induced CAR signalling, but also signalling via the TCR of the γδ T cell, leading to a 2-way synapse with each cell potentially both target and effector. When normal γδ T outnumbered anti-γδ CAR-T, the balance of cytotoxicity resulted in anti-γδ CAR-T cell lysis and expansion of the normal cells. The potential clinical consequences for anti-γδ TCR CAR therapy are unclear and would be difficult to ascertain pre-clinically due to a lack of relevant immunocompetent models. However, in patients receiving anti-γδ CAR-T, it is likely that the CAR-T: normal γδ T cell ratio at the tumour site would be high in the critical initial CAR-T expansion phase, following lymphodepleting chemotherapy. Here, we have demonstrated the feasibility of engineering normal αβ T cells to express anti-γδ TCR CAR and have shown that anti-γδ TCR CAR-T cells can specifically kill malignant γδ cells both in vitro and in vivo. Our approach offers the first proposed strategy to bring highly potent CAR-T cells to the treatment of γδ T cell malignancies, where there is a major unmet need for effective therapies. Clinical assessment of this approach is warranted.

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